(4) Ethanol production by thermotolerant, fermentative yeasts Strains with upper temperature limits for fermenta-
tion above 42℃ were selected and used to prepare seed cul- tures. Each strain was inoculated into a test tube containing 10 mL of YPD liquid medium and cultured at 37℃ for 8 to 10 h by reciprocal shaking at 120 rpm. A 200-µL volume of the culture was re-inoculated into a 300-mL shaking flask containing 100 mL YPD medium and cultured at 37℃ to the early stationary phase. The culture was then centrifuged at 3000 × g for 5 min at 4℃, washed twice and resuspended in sterile tap water containing 0.1% glucose. A 100-mL volume of Tyndalized (Vaughan-Martini and Martini, 1998) YPD screening medium containing 15% glucose was dispensed into a 300-mL Erlenmeyer flask and inoculated with 5 g of wet seed culture. The fermentation flasks were fitted with stoppers to vent CO2 through a water trap and incubated in a Bio Shaker BR-15LF incubator at 40℃ with shaking (130 rpm). A 10-mL sample was withdrawn from each flask after 48 h and centrifuged at 6000 × g for 10 min at 4℃ to remove cells. The supernatants were used for ethanol determination as described by Suresh et al. (1999).